Natural cheese traditionally is made by developing acidity in milk and setting the milk with a clotting agent, such as rennet, or by developing acidity to the isoelectric point of the protein. The set milk is cut and whey is separated from the curd. The curd may be pressed to provide a cheese block. Curing typically takes place over a lengthy period of time under controlled conditions. Cheddar cheese, for example, is often cured for a number of months or even longer, to obtain the full flavor desired.
Numerous reports have been published implicating several compounds to be important in the development of cheese flavor in cheese products. The main classes of compounds thought to contribute to flavor generation in cheese include amino acids, peptides, carbonyl compounds, fatty acids, and sulfur compounds. Urbach, G., “Contribution of Lactic Acid Bacteria to Flavor Compound Formation in Dairy Products,” Int'l Dairy J., 1995, 3:389-422. Several volatile compounds including fatty acids, esters, aldehydes, alcohols, ketones, and sulfur compounds are included in lists describing the aroma of various cheeses. Production of several of these aroma and flavor compounds have been attributed to multiple enzymatic reactions and/or chemical reactions that take place in a sequential manner in ripening cheese.
Various microorganisms have been identified and selected for their ability to produce particular flavors in a cheese-ripening environment. These flavors arise through a series of enzymatic steps. For example, in cheese, degradation of proteins by proteases and peptidases can lead to the production of peptides and free amino acids. These precursors are shuttled through subsequent enzymatic reactions resulting in the formation of flavor compounds. An understanding of these reactions helps in the creation of flavors of a desired cheese type. Fox, P., Cheese: Chemistry, Physics and Microbiology, pp. 389-483, 1993.
The role of amino acid catabolism in the development of cheese aroma and flavor has been identified to be a rate limiting step in the development of cheese flavors. Yvon et al., “Cheese flavour formation by amino acid catabolism,” Int. Dairy J. 11 (2001) 185-201. α-Keto acids are generally recognized as a key intermediate in the metabolism and interconversion of amino acids. Some of the main pathways identified in lactic acid bacteria include transamination reactions catalyzed by aminotransferases. They are responsible for the deamination of amino acids and formation of keto acids. A disadvantage with the aminotransferase enzyme is that it requires the presence of an amino group acceptor, which is limiting in the cheese matrix and needs to be supplemented to enhance transamination. According to the literature, the creation of aroma compounds and cheese flavor is greatly enhanced with the addition of α-ketoglutarate, an amino group acceptor. Yvon et al., “Adding α-Ketoglutarate to Semi-hard Cheese Curd Highly Enhances the Conversion of Amino Acids to Aroma Compounds,” Int. Dairy J. 8 (1998) 889-898.
The literature also describes acceleration of the development of flavor compounds by the exogenous addition of enzymes and cell extracts (e.g., U.S. Pat. No. 6,649,199), and by the supplementation of a cheese matrix with intermediates of amino acid catabolism (e.g., U.S. Pat. No. 6,586,025; Banks et al., “Enhancement of amino acid catabolism in Cheddar cheese using α-ketoglutarate . . . ,” Int. Dairy J. 11 (2001) 235-243).
According to at least one literature reference, D-amino acid oxidase is a flavoprotein which deaminates D-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide (H2O2) in the presence of molecular oxygen; the resulting hydrogen peroxide is degraded into water and molecular oxygen in the presence of catalase whereby keto acids remain as the final product. Upadhya et al., “D-Amino Acid oxidase and catalase of detergent permeabilized Rhodotorula gracilus cells and its potential use for the synthesis of α-keto acids,” Process Biochem., 35 (1999) 7-13. U.S. Pat. No. 6,461,841 describes an isolated L-amino acid oxidase from Rhodococcus species encoded by a DNA molecule hybridizing to a particular nucleic acid sequence, which can be used for contacting an L-amino acid for the production of a keto acid. Neither of these last two-mentioned literature references refer to cheese microorganisms or a cheese-making environment involving such organisms. It has been reported that, although deamination of amino acids to α-keto acids might be catalyzed by either dehydrogenases or oxidases, such activities towards aromatic and branched-chain amino acids and methionine have never previously been detected in cheese microorganisms. Yvonet al., “Cheese flavour formation by amino acid catabolism,” Int. Dairy J. 11 (2001) 185-201, 189-190.
Cheese manufacturers are interested in developing cheese products requiring less storage time before they are ripe enough for commercial distribution. Cheese makers have used a wide variety of different techniques in efforts to accelerate the cheese curing or ripening process. U.S. Pat. No. 6,649,200 provides a summary of a number of these techniques used for accelerating ripening of hard block cheeses.
Another approach used to avoid lengthy cheese ripening periods has been to make a cultured cheese concentrate (“CCC”) having more intense cheese flavor, and then use it as a cheese flavoring agent in another bulk material. CCC's have been manufactured that attain full cheese flavor development within a number of days instead of months. These CCC's are added to other bulk foods, such as process cheeses or snack foods, to impart or intensify a cheese flavor. Methods for the manufacture of such cheese-flavored concentrates have been described in U.S. Pat. No. 4,708,876. Typically the process involves a dairy substrate that is cultured with a lactic culture followed by addition of various proteases, peptidases, and lipases. U.S. Pat. No. 4,708,876 describes cheese flavored concentrates that can be obtained from milk as a starting material, instead of cheese curds, or without formation of whey by-product. U.S. Pat. No. 6,214,586 describes use of live cultures having high levels of proteolytic enzymes and peptidolytic enzymes to debitter enzymatic modified cultures (EMC's).
Methods of cream cheese manufacture have been previously described in publications such as by Kosikowski and Mistry in Cheese and Fermented Milk Foods, 3rd Ed.
Although these prior processes may produce an accelerated development, or an enhancement, of cheese flavor, they do not produce enhancements that target specific cheese flavor components. More recently a technology has been developed to produce a natural biogenerated cheese flavoring system that can be used to prepare different cheese products/derivatives, targeted at various cheese flavor profiles using a modular approach to flavor creation, which is described in, for example, U.S. Pat. No. 6,406,724. The cheese flavoring system described in this patent is derived from different components, wherein the individual components are combined in different ratios to provide specific flavor profiles in the cultured cheese concentrate products.
Despite the developments described in the above publications, a need still exists for alternative routes for making cheese flavoring systems, especially those produced via natural processes.